The Role And Mechanism Of VASN In Helicobacter Pylori Pathogenesis And Gastric Carcinogenesis

Background and objective:Helicobacter pylori(Hp)infection is the initiating factor in the evolution of chronic gastritis?atrophic gastritis?intestinal metaplasia?dysplasia?gastric cancer.It is designated as a Class I carcinogen by the International Agency for Research on Cancer(IARC),but its pathogenic mechanism is currently unclear.Therefore,finding the key driver molecules in the pathogenesis of Hp and elucidating their roles and mechanisms in the occurrence and development of gastric cancer is extremely important for the effective prevention and treatment of Hp-related gastric cancer.The preliminary transcriptomics research of our group found that Hp and its main virulence factor cytotoxin-related gene A(Cag A)can up-regulate the expression of Vasorin(VASN)in gastric epithelial cells.Further bioinformatics analysis found that VASN expression is significantly higher than that of adjacent tissues,and the prognosis of gastric cancer patients with high VASN expression was worse than that of patients with low VASN expression,suggesting that VASN may play a role in gastric cancer and affect the prognosis of gastric cancer patients.VASN is a type I transmembrane glycoprotein.It is highly expressed in glioblastoma,prostate cancer,lung cancer and other tumor cells.It has pro-proliferation and anti-apoptotic effects,and is related to tumor invasion and poor prognosis.Therefore,we speculate that VASN may be a key molecule of Hp-induced gastric mucosal carcinogenesis,and can promote the occurrence and development of gastric cancer.However,no studies have reported the role of VASN in the occurrence and development of gastric cancer and the pathogenesis of Hp.The relationship between VASN and gastric cancer and Hp infection,as well as the underlying mechanism remains to be elucidated.Therefore,this article aims to explore the relationship between VASN and Hp induced gastric carcinogenesis,as well as the molecular functions and underlying mechanisms in gastric cancer,which provided experimental and theoretical basis for the prevention and molecular therapy of gastric cancer.Materials and Methods:Screening and identifying the core gene VASN in gastric mucosal epithelial cell lesions caused by Hp infection1.Screening the core genes in Hp and Cag A induced gastric mucosal epithelial cell lesions by Transcriptomic analysis:(1)Construct GES-1 cell models infected with7.13 wild-type and?cag A Hp strains in vitro,total cell RNA was extracted for RNA sequencing analysis,and gene expression differential analysis was performed;(2)q RT-PCR was used to verify the core genes and selected the most critical core genes that have been verified for follow-up research.2.Cell-level verification of the effects of Hp and Cag A on VASN protein expression:(1)Different Hp wild-type strains including 7.13,PMSS1,ATCC43504,G27 and CCS9803 infected GES-1 cells,respectively;(2)7.13 and PMSS1 Infected GES-1 cells and AGS cells with different multiplicity of infection(MOI)respectively to determine the optimal MOI;(3)The wild-type and?cag A strains of 7.13 and PMSS1 were used to infect GES-1 cells and AGS cells with the optimal MOI respectively;Western blot was used to detect the expression level of VASN.3.the effect of Hp infection on VASN expression was verified in vivo:C57BL/6mouse model of Hp infenction was constructed,and Western blot was used to detect the expression of VASN in mouse gastric mucosa.4.The expression of VASN in the process of gastric mucosal lesions in clinical patients and its relationship with Hp infection:Hp-positive and Hp-negative gastric mucosal lesions were collected separately,including mild non-atrophic gastritis,severe non-atrophic gastritis,intestinal metaplasia and gastric cancer,and VASN expression was detected by immunohistochemistry.The expression of VASN in gastric cancer tissues and its relationship with the prognosis of gastric cancer patients1.The expression of VASN in gastric cancer:(1)Using the Oncomine database to analyze the expression of VASN in different tumors,and the expression of VASN in different Lauren types of gastric cancer;(2)Using the GEO database to analyze the expression of VASN in normal gastric mucosal tissue and gastric cancer,as well as its expression level in adjacent tissues;(3)Collecting gastric cancer and adjacent tissues,and detecting the expression level of VASN by immunohistochemistry and Western blot.2.The relationship between VASN and the prognosis of patients with gastric cancer:the relationship between the expression level of VASN and the prognosis of patients with gastric cancer was analyzed in Kaplan-Meier plotter database.Effect of VASN on the biological phenotype of gastric epithelial cells and gastric cancer cells1.Construction of stable transgenic cell lines with high VASN expression and low VASN expression:GES-1 cells with high VASN expression(VASN^highGES-1)and GES-1cells with low VASN expression(VASN^lowGES-1),as well as AGS cells with high VASN expresson were constructed by lentiviral methods.2.The effect of VASN on the proliferation ability of gastric epithelial cells and gastric cancer cells:the proliferation ability of VASN^highGES-1,VASN^lowGES-1 and VASN^highAGS and their corresponding WT cells were detected by Real-Time Cell Analyzer(RTCA)real-time analysis,CCK8,and clone formation,respectively.3.The effect of VASN on the migration and invasion ability of gastric epithelial cells and gastric cancer cells:transwell experiments were used to detect the migration and invasion ability of VASN^highGES-1,VASN^lowGES-1 and VASN^highAGS and their corresponding WT cells.The biological functions of VASN revealed by Transcriptomic and proteomic analysis1.The effects of VASN on gastric epithelial cells and gastric cancer cells at the transcriptional level:(1)Transcriptomics analyses:the total RNA of VASN^highGES-1,VASN^lowGES-1and VASN^high AGS,as well as their corresponding WT cells were extracted.After Oligo d T,enriched,fragmented,c DNA reversely synthesized,fragment screening and library enrichment,and finally through Illumina Hi Seq Xten/Nova Seq6000 online sequencing;(2)Differential expression analysis:differential gene analysis software:DESeq2,screening threshold:difference multiple?2 or?-2,FDR<0.05.(3)Bioinformatics analysis:GO and KEGG enrichment analysis of differential genes.2.The effect of VASN on the protein level of gastric epithelial cells and gastric cancer cells:(1)Proteomic analysis:extract the total proteins of VASN^highGES-1,VASN^lowGES-1 and VASN^highAGS and their corresponding WT cells and perform protein quantitative analysis,then undergo enzymatic alkylation and labeling,RPLC one-dimensional separation,Tandem mass spectrometry analysis,and finally a protein search.The differential gene analysis software is:DESeq2,and the screening threshold is:multiple of difference?1.2 or?0.83,FDR<0.05;(3)Bioinformatics analysis:GO and KEGG enrichment analysis of differential proteins.VASN regulates the biological changes of gastric epithelial cells and gastric cancer cells through the PI3K/AKT signaling pathwayThe above-mentioned omics studies have found that VASN can regulate the PI3K/AKT signaling pathway,and follow-up studies will focus on this signaling pathway.1.Correlation analysis of key molecules in VASN and PI3K/AKT signaling pathway:the correlation between VASN and AKT1,AKT2,AKT3 in gastric cancer were analyzed in TIMER database.2.The regulation of VASN on PI3K/AKT signaling pathway:Western blot was used to detect the expression levels of the related molecules in PI3K/AKT signaling in VASN^highGES-1,VASN^lowGES-1 and VASN^highAGS cells.3.The effect of PI3K/AKT inhibitor on the proliferation of VASN:After VASN^highAGS administered PI3K/AKT inhibitor MK-2206,real-time label-free cell analysis and CCK8 method were used to detect cell proliferation.VASN^highGES-1 and VASN^highAGS cells were given 0(DMSO treatment group),1?M and 3?M AKT inhibitor MK-2206,and then at 6h,24h,48h,72h and 96h,the cell proliferation ability of VASN^highGES-1 and VASN^highAGS cells were detected by CCK8 method;When VASN^highAGS cells were cultured with 1?M MK-2206,Real-Time Cell Analyzer instrument was used to detect the proliferation level of cells at 0-96h.4.The effect of VASN on PI3K/AKT signal network:PCR array was performed to detect the expression changes of 84 signal molecules in the PI3K/AKT signal network of VASNlow GES-1 cells.Results:Screening and identifying the core gene VASN of gastric mucosal epithelial cell lesions caused by Hp infection1.Transcriptomics screening of Hp and Cag A-induced changes in the core genes of gastric epithelial cells:A total of 28 core differential genes(P<0.05)with significant changes in expression levels after Hp infection and dependent on its virulence factor Cag A were screened.The top 11 genes with the most significant differences including VASN were selected for q PCR verification,and it was found that Hp infection up-regulated the expression of VASN and relied on Cag A.2.Cellular and animal levels to verify the effect of Hp infection and Cag A on the expression of the core gene VASN protein:(1)Different Hp strains(7.13,PMSS1,ATCC43504,G27,CCS9803)infected GES-1 cells significantly increased VASN expression;(2)When 7.13 Hp strains and PMSS1 Hp strains of different MOI infect gastric epithelial cells and gastric cancer cells,whether in GES-1 cells or AGS cells,the expression of VASN gradually increased with the increase of MOI,and the expression of VASN was up-regulated when MOI=100 More significant;(3)7.13Hp?cag A and PMSS1Hp?cag A strains infected GES-1 and AGS cells for 6h,the expression of VASN was lower than the corresponding Hp WT strain;(4)The expression of VASN in the gastric mucosal tissue of C57BL/6 mice was increased after infection with Hp SS1 strain.3.The expression of VASN in the process of gastric mucosal lesions in clinical patients and its relationship with Hp infection:(1)VASN is expressed in gastric mucosal gland cells and inflammatory cells,and it is significantly lower in the mild non-atrophic gastritis group than in the other groups(P<0.05);(2)In each pathological stage,whether it is glandular cells or inflammatory cells,the expression level of VASN in Hp-positive patients was significantly higher than that of Hp-negative patients(P<0.05);(3)In gland cells,whether Hp-infected or uninfected,The expression of VASN in gastric cancer was higher than that of other groups(P<0.05),and the expression of severe non-atrophic gastritis and intestinal metaplasia in Hp-uninfected patients was also significantly higher than that of mild non-atrophic gastritis(P<0.05).There was no statistically significant difference in VASN expression among Hp-infected patients with mild non-atrophic gastritis,severe non-atrophic gastritis,and intestinal metaplasia(P>0.05);(4)In inflammatory cells,VASN expression in Hp-uninfected patients was in mild The non-atrophic gastritis group was significantly lower than the severe non-atrophic gastritis and intestinal metaplasia group(P<0.05).The VASN expression of Hp infection in the mild non-atrophic gastritis group was significantly lower than that in the severe non-atrophic gastritis and gastric cancer group(P<0.05).The expression of VASN in gastric cancer and its relationship with the prognosis of gastric cancer patients1.The expression of VASN in gastric cancer:(1)Analysis in the Oncomine database shows that VASN is abnormally expressed in multiple tumors and is highly expressed in gastric cancer;in addition,VASN has different Lauren scores in diffuse gastric cancer,intestinal gastric cancer,and mixed gastric cancer Significantly high expression in type gastric cancer(P<0.05);(2)Analysis in the GEO database showed that the expression of VASN in gastric cancer was significantly higher than that of normal gastric mucosa(P<0.01)and adjacent tissues((P<0.01));(3)IHC results showed that whether in gastric mucosal gland cells or inflammatory cells,the expression of VASN in gastric cancer tissue was significantly higher than that in adjacent tissues((P<0.01);Western blot results showed that the expression of VASN in gastric cancer tissue was significantly higher than that in adjacent tissues.2.The relationship between VASN and the prognosis of gastric cancer patients:The overall survival(OS),first progression survival(FP)and post progression survival(PPS)of gastric cancer patients with high expression levels of VASN are significantly shorter than Gastric cancer patients with low VASN expression(P<0.001).Effect of VASN on the biological phenotype of gastric epithelial cells and gastric cancer cells1.The effect of VASN on the proliferation,migration and invasion ability of gastric epithelial cells:(1)Real-Time Cell Analyzer(RTCA)real-time analysis and CCK8 results showed that the proliferation ability of VASN^highGES-1 was significantly higher than that of WT-type GES-1(P<0.05),while VASN^lowGES-1proliferation ability was significantly lower than WT type GES-1((P<0.05);(2)The results of colony formation assay showed that the number of clone formation of VASN^highGES-1 was significantly higher than that of WT type GES-1(P<0.001),while VASNlow GES-1 The number of clone formation of WT was significantly lower than that of WT GES-1(P<0.001).(3)Cell migration and invasion were assessed by Transwell experiments and the results showed that the migration and invasion ability of VASN^highGES-1 was significantly higher than that of WT-type GES-1(P<0.05),while the migration and invasion ability of VASN^lowGES-1 is significantly lower than that of WT-type GES-1(P<0.05).2.The effect of VASN on the proliferation,migration and invasion of gastric cancer cells:(1)The results of RTCA real-time analysis and CCK8 showed that the proliferation ability of VASN^highAGS was significantly higher than that of WT-type AGS(P<0.05);(2)The results of colony formation assay showed that The number of clone formation of VASN^highAGS was significantly higher than that of WT AGS(P<0.01).(3)The results of transwell experiments showed that the migration and invasion ability of VASN^highAGS was significantly higher than that of WT-type AGS(P<0.05)The biological functions of VASN revealed by Transcriptomic and proteomic analysis1.The effect of VASN on the transcription level of gastric epithelial cells and gastric cancer cells.(1)The effect of VASN on the transcription level of gastric epithelial cells.(1)The effect of VASN overexpression on the transcription level of gastric epithelial cells:i.statistics of differentially expressed genes(DEGs):A total of 419DEGs,including 190 up-regulated genes and 229 down-regulated genes;ii.GO enrichment analysis of DEGs:DEGs mainly enriched to 33 cellular components,28molecular functions and 405 biological processes(P<0.05,FDR<0.05);Cellular components mainly included cytoplasm and cell membrane;molecular functions mainly included protein binding and signal receptor binding;biological processes mainly included cell proliferation and differentiation,Inflammatory immune response and so on;iii.KEGG enrichment analysis of DEGs:DEGs are mainly enriched in 51signal pathways(P<0.1),including Epithelial cell signaling in Helicobacter pylori infection,and NF-?B signal pathway,TLR-like receptor signaling pathway,TGF-?signaling pathway,MAPK signaling pathway,IL-17 signaling pathway,Jak-STAT signaling pathway,PI3K/AKT signaling pathway,etc.(2)The effect of VASN low expression on the transcription level of gastric epithelial cells:i.statistics of DEGs:A total of 573 DEGs,including 403 up-regulated genes and 170 down-regulated genes;ii.GO enrichment analysis of DEGs:DEGs significantly enriched to 67 cellular components,41 molecular functions and 461biological processes.Cellular components mainly include cell membrane,cytoplasm and extracellular regions;molecular functions mainly include binding,signal receptor binding and cytokine activity;biological processes mainly include cells proliferation,differentiation,regulation of cell migration,inflammatory response,cell surface receptor signaling pathways,etc.iii.KEGG enrichment analysis of DEGs:DEGs are mainly enriched in 54 signal pathways(P<0.1),including cytokine-cytokine receptor signaling pathway,NOD-like receptor signaling pathway,IL-17 signaling pathway,Toll-like receptor signaling pathway,NF-?B signaling pathway,epithelial cell signaling in Helicobacter pylori infection,PI3K-AKT signaling pathway,etc.(3)The common signaling pathways of KEGG enrichment of DEGs with VASN overexpression and knockdown in gastric epithelial cells:there were 31 signaling pathways including TNF signal pathway,cytokine-cytokine receptor interaction,IL-17 signaling pathway,NF-?B signaling pathway,epithelial cell signaling in Helicobacter pylori infection,Toll-like receptor signaling pathway,MAPK signaling pathway,Jak-STAT signaling pathway and PI3K-AKTsignaling pathway(P<0.1).(2)The effect of VASN on gastric cancer cells at transcription level.The effect of VASN overexpression on gastric cancer cells at transcription level:(1)Statistics of DEG:There are 186 DEGs,of which 158 are up-regulated genes and28 are down-regulated genes;(2)The GO enrichment analysis of DEGs:DEGs enriched to 7 Cellular components,10 molecular functions and 53 biological processes.Cellular components mainly included extracellular regions,cytoplasm,and intrinsic components of plasma membrane.Molecular functions mainly included carboxylic acid transmembrane transport activity and protein binding.Biological processes mainly included anti-tumor drug response,signal transduction,inflammatory response,etc.(3)KEGG enrichment analysis of DEGs:DEGs mainly enriched to 35 signaling pathways(P<0.1),including cytokine-cytokine receptor interaction,PI3K/AKT signaling pathway,HIF-1 signaling pathway,AMPK signaling pathway,focal adhesion,leukocyte transendothelial migration,etc.(3)The signaling pathways that VASN affects gastric epithelial cells and gastric cancer cells at the transcriptional level:there are 6 common signaling pathways mainly included mucin type O-glycan biosynthesis,cytokine-cytokine receptor interactions,human cytomegalovirus infection,PI3K-Akt signaling pathway,Cytosolic DNA-sensing pathway and relaxin signaling pathway(P<0.1).2.The effect of VASN on gastric epithelial cells and gastric cancer cells at protein level.(1)The effect of VASN on gastric epithelial cells at protein level.(1)The effect of VASN overexpression on gastric epithelial cells at the protein level:i.the statistics of differentially expressed proteins(DEPs):a total of 790 DEPs,including 367 up-regulated proteins and 423 down-regulated proteins;ii.GO enrichment analysis of DEPs:DEPs significantly enriched to 45 kinds of cell components,97 kinds of molecular functions and 584 kinds of biological processes.Cellular components mainly included exosomes,extracellular matrix and anchoring connections.The molecular functions mainly included signal receptor activity,protein binding and cell adhesion molecule binding,etc.The biological process mainly included the regulation of cell proliferation and cell adhesion,regulation and inflammatory response,etc.;iii.KEGG enrichment analysis of DEPs:DEPs mainly enriched to 44 signaling pathways(P<0.1),including ECM receptor interaction,HIF-1 signaling pathway,cytokine-cytokine receptor interaction,Wnt signaling pathway and PI3K/AKT signaling pathway,etc.(2)The effect of VASN low expression on gastric epithelial cells at protein level:i.DEG statistics:A total of 1223 DEGs,including 579 up-regulated proteins and 644down-regulated proteins;ii.GO enrichment analysis of DEGs:DEGs significantly enriched to 64 cell components,42 molecular functions and 207 biological processes.Cell components mainly included extracellular matrix,plasma membrane and cytoskeleton.Molecular functions mainly included cell adhesion molecule binding,extracellular matrix binding and drug binding.Biological processes mainly included cell differentiation,cell migration and acute inflammation.Iii.KEGG enrichment analysis of DEGs:mainly enriched to 33 signal pathways(P<0.1),including ECM receptor interaction,focal adhesion,antigen processing and presentation,metabolic pathways,cell adhesion molecules,cancer pathways,etc.(3)The common signaling pathways of KEGG enrichment of DEPs with VASN overexpression and knockdown in gastric epithelial cells:There are 13 common signaling pathways,including ECM receptor interaction,metabolic pathway,cancer pathway,cell adhesion molecule and WNT Signal pathways,etc.(P<0.1).(2)The effect of VASN on gastric cancer cells at the protein level.The effect of VASN overexpression on gastric cancer cells at the protein level:(1)DEP statistics:A total of 915 DEPs,including 421 up-regulated proteins and 494down-regulated proteins.(2)GO enrichment analysis of DEPs:50 cell components,56 molecular functions,and 331 biological processes are significantly enriched.Among them,the cell components mainly included the endoplasmic reticulum cavity,extracellular vesicles,and extracellular regions,etc.Molecular functions mainly included cell adhesion molecule binding,cyclin-dependent serine/threonine kinase regulatory activity and cell-cell adhesion mediator activity,etc.Biological processes mainly included biological adhesion,cell migration,and regulation of epithelial cell apoptosis,positive regulation of inflammatory response,etc.(3)KEGG enrichment analysis of DEPs:mainly enriched to 47 signal pathways(P<0.1),including metabolic pathways,cell adhesion molecules and ECM receptor interactions,etc.(3)The signaling pathways that VASN affects gastric epithelial cells and gastric cancer cells at the protein level:there are 5 common signaling pathways,included ECM-receptor interactions,metabolic pathways,carbon metabolism,cell adhesion molecules and biosynthesis of amino acids.VASN regulates the biological changes of gastric epithelial cells and gastric cancer cells through the PI3K/AKT signaling pathway.The above-mentioned omics studies have found that VASN can regulate the PI3K/AKT signaling pathway,and follow-up studies will focus on this signaling pathway.1.Correlation analysis between VASN and key molecules of PI3K/AKT signaling pathways:the analysis results in TIMER database showed that VASN and AKT1(P=1.03e-11,r=0.328),AKT2(P=0e-0,r=0.426)and AKT3(P=9.01e-31,r=0.525)were significantly correlated.2.VASN regulates the PI3K/AKT signaling pathway in gastric epithelial cells and gastric cancer cells:(1)the phosphorylation levels of PI3K and AKT in VASN^highGES-1 were higher than those of WT-type GES-1,while the phosphorylation levels of PI3K and AKT in VASN^lowGES-1 was significantly lower than that of WT-type GES-1;(2)The phosphorylation level of PI3K and AKT in VASN^highAGS was significantly higher than that of WT-type GES-1.3.The effect of AKT inhibitor on the proliferation of VASN:After treating VASN^highGES-1 cells and VASN^highAGS cells with different concentrations of MK-2206,the cell proliferation ability was significantly reduced(P<0.05),and with the increase of MK-2206 concentration,the cell proliferation ability was significantly reduced.4.The effect of VASN on the PI3K/AKT signal network:PCR array results showed that 72 of the 84 signal molecules in the PI3K/AKT signal network had significant changes after VASN is knocked down(P<0.05),of which 71 genes was down-regulated,such as FOXO1,ELK1,CHUK,APC,ADAR,RBL2,PIK3CA,PIK3R1,CDKN1B,CCND1(P<0.05);the expression of another gene,GRB10,was up-regulated(P<0.05).Conclusions:1.Hp infection can up-regulate the expression of VASN,which depends on its virulence factor Cag A.2.VASN is related to the Hp-induced gastric caecinogedesis.3.VASN overexpression in gastric cancer is related to the poor prognosis of gastric cancer patients.4.VASN promotes the proliferation,migration and invasion of gastric epithelial cells and gastric cancer cells.5.VASN has effects on gastric epithelial cells and gastric cancer cells at both the transcription level and protein level.They are involved in cell differentiation,proliferation,apoptosis and migration and other biological processes related to cell growth and death,as well as inflammatory immunity and signal transduction that related to biological processes.In addition,it affects multiple tumor and inflammatory immune-related signaling pathways,which may be the molecular basis for its role in the occurrence and development of gastric cancer and the Hp pathogenesis.6.VASN may exert its biological function through PI3K/AKT signaling pathway.Blocking AKT signaling can inhibit the proliferation ability of VASN.